Epidermal differentiation is essential for survival and impaired in common skin diseases, including psoriasis. Keratinocytes interact and communicate with their neighbors via cell surface proteins; this helps determine whether epidermal cells divide in the basal layer, differentiate in the suprabasal layers and die in the cornified layer. Te long-term goal of this proposal is to define the role of a cell-cell communication pathway, termed the EphA/ephrin-A signaling axis, in epidermal [differentiation] and disease. This large family of receptor tyrosine kinases plays a key role in embryonic development and cancer progression but has not been thoroughly investigated in normal adult epithelial tissues, especially the skin. Species differences in the character and molecular composition of EphA receptors in the epidermis necessitate the application of experimental approaches that take advantage of human cells. Consequently, an organotypic raft model of human epidermis that has been adapted for long-term gene silencing and reconstitution will be employed to define the reciprocal roles for EphA1 and EphA2 in epidermal differentiation and morphogenesis. We will test the innovative hypothesis that EphA1 and EphA2 exhibit discrete functions in keratinocyte differentiation that are controlled by their responses to ephrin ligand binding. In the first aim, the mechanism(s) by which EphA2 maintains keratinocytes in an undifferentiated state until this receptor is engaged and downregulated by ephrin-A1 will be elucidated. The importance of the EphA2 cytoplasmic domain in inhibiting keratinocyte differentiation [by interacting with SHIP2, a modulator of the EGFR signaling pathway,] will be determined using genetic approaches that remove key signaling elements within this region. The response of EphA2 to ligand binding will be defined using gene mutagenesis approaches [that focus on the downstream activation of Notch signaling to promote differentiation.] The second aim will test the novel hypothesis that EphA1 promotes keratinocyte differentiation in response to ephrin-A1 binding only after EphA2 is lost from the cell surface. Mutating and replacing the extracellular and cytoplasmic subdomains of EphA1 with EphA2 sequences will reveal the key signaling elements that trigger keratinocyte differentiation, focusing on the ability of the EphA1 SAM domain to regulate the integrin-linked kinase-RhoA-[serum response factor] pathway. [The translational potential of the proposal is strengthened by the ability to harness the epidermal EphA/ephrin-A axis in inflammatory skin conditions where their relative expression changes and keratinocyte differentiation is impaired.] Important interactions with local (Northwestern U.-Skin Disease Research Center, Robert M. Lavker; [Northwestern U.-Proteomics Core, Dhaval Nanavati]) and outside (Bing-Cheng Wang, Case Western Reserve; [G.-Paolo Dotto; Harvard; Lina Dagnino; Western U.]) groups studying epithelial biology and Eph/ephrin signaling enhance the likelihood of success and impact of the proposal.